Hepatic fibrosis results from chronic liver injury induced by alcohol abuse, hepatitis and other causes, progressing to cirrhosis and liver dysfunction. Recent studies have focused on the role of hepatic stellate cells (HSC), the major source of fibrillar collagen in hepatic fibrosis and cirrhosis. Normally, HSC display a quiescent phenotype, and function to store lipids in the form of retinoids. With liver injury, HSC lose their retinoid stores and adopt an activated phenotype, characterized by increased collagen secretion. A major factor in HSC activation is the profibrotic cytokine transforming growth factor-2 (TGF2). A second major factor is the loss of expression of the peroxisome proliferator-activated receptor 3 (PPAR3). The presence of PPAR3 is critical for maintenance of the quiescent, adipogenic phenotype of HSC. Therefore, targeted inhibition of TGF2 signaling, and activation of PPAR3, are promising approaches to the treatment of fibrotic liver diseases. Our work has focused on the hormone relaxin and its antifibrotic effects in the liver. Relaxin inhibits the fibrotic phenotype of HSC, and decreases collagen deposition in models of liver fibrosis in vivo. In extrahepatic fibroblastic cells, the mechanism of relaxin's antifibrotic effects was through inhibition of TGF2 signaling, but at present little is known about the mechanism of relaxin inhibition of the fibrotic phenotype of HSC. Preliminary data suggests that relaxin inhibits TGF2 signaling in HSC through a mechanism involving the downstream mediators of TGF2 signaling, the proteins Smad2 and Smad3. Furthermore, we have shown that relaxin activates PPAR3, but the signaling mechanisms for this effect are unknown. Consistent with these findings, relaxin treatment increased the response to a PPAR3 agonist in the treatment of established hepatic fibrosis in vivo. The objective of this proposal is to determine the mechanisms of the antifibrotic actions of relaxin by testing the hypothesis that relaxin antagonizes TGF2 signaling and upregulates PPAR3 activity. To test this hypothesis, three Specific Aims are proposed: Specific Aim #1. Determine the mechanism of relaxin antagonism of TGF2 signaling in hepatic stellate cells. The working hypothesis is that relaxin activation of adenylyl cyclase and activation of downstream pathways results in perturbation of Smad2 and Smad3 phosphorylation and nuclear translocation. Specific Aim #2. Determine the mechanism of PPAR3 activation by relaxin in hepatic stellate cells. Based on preliminary data, the working hypothesis is that relaxin signaling activates PPAR3 in a ligand- independent manner. Specific Aim #3. Determine the therapeutic efficacy of using relaxin in combination with PPAR3 agonists to treat extensive fibrosis and cirrhosis. The working hypothesis is that relaxin will increase the response to PPAR3 ligands in mouse models of established hepatic fibrosis and cirrhosis. To achieve Aim 1, hepatic stellate cells will be used determine the mechanism for relaxin inhibition of TGF2 signaling. This will be achieved by using specific downstream activators and siRNA approaches to manipulate signaling pathways. For Aim 2, reporter constructs in conjunction with coprecipitation and signaling pathway manipulations will be used to identify the mechanism of PPAR3 activation by relaxin. Finally, for Aim 3, mouse models of established fibrosis and recovery from cirrhosis will be used to determine the effect of the combined treatment of relaxin and the PPAR3 ligand pioglitazone. The proposed research is significant, because understanding the mechanism for relaxin on the TGF2 and PPAR3 pathways will allow the development of new approaches to manipulate these signaling pathways. The research proposed in this application may lead to targeted therapeutic strategies for the treatment of liver disease, a major health concern in the United States. PUBLIC HEALTH RELEVANCE: Potential Impact on Veterans Health Care: Cirrhosis affects 900,000 people and causes 36,000 deaths annually in the U.S. The major causes of cirrhosis (hepatitis C virus and alcohol abuse) are significantly more prevalent in Veterans than in the general population, and therefore treatment of fibrosis and cirrhosis an area of particular concern in the VA Health Care System. Treatment options are currently limited to removal of the cause of the injury, which is not always possible or effective, or transplantation. The research proposed in this application may lead to targeted therapeutic strategies for the treatment of liver disease, a major health concern in the Veteran population.